Custom CRISPR solutions

Don’t see what you’re looking for? We are continually expanding our CRISPR product line, and we may have what you need. If you are interested in custom libraries, other CRISPR enzymes, formulations, or other CRISPR tools contact our CRISPR experts today to discuss customized solutions for your research: CRISPR@idtdna.com.

Alt-R S.p. Cas9 nickases

Cas9 nickases allow specific cutting of only one strand at the DNA target site. Cuts to both strands of DNA are accomplished by using either Alt-R S.p. Cas9 D10A Nickase V3 or Alt-R S.p. Cas9 H840A Nickase V3, with 2 gRNAs that target two neighboring Cas9 sites, one on either strand of the target region. This functionally increases the length of the recognition sequence from 20 to 40 bases. For more information about using Cas9 nickases, see the application note.

Alt-R S.p. dCas9 protein

Alt-R S.p. dCas9 Protein V3 has mutations that result in the loss of nuclease activity. This protein can form RNP complexes with Alt-R gRNAs and bind to the target region specified by the gRNA without cutting the DNA.

Attention: Unlike S. pyogenes Cas9, which cleaves most NGG PAM sites to some degree, some of the tested TTTV sites show no cleavage by A.s. Cas12a nuclease. We recommend using positive control crRNAs to establish that your cells can be edited by Cas12a. In addition, we suggest testing 3 or more crRNAs per target gene.

Comparison of CRISPR genome editing using Cas9 vs. Cas12a (Cpf1)

Cas9 system

Cas12a system

Applications

General genome editing

For species with AT-rich genomes

For regions with limiting design space for use of the CRISPR-Cas9 system

Improved editing efficiency using Alt-R S.p. Cas9 Nuclease V3

Alt-R S.p. Cas9 Nuclease V3 is designed to maximize the efficiency of genome editing across a broad number of sites. Modification of the expression construct facilitates nucleus-targeted delivery, resulting in enhanced cleavage, particularly at difficult targets (Figure 1).

Potent editing with the Alt-R S.p. Cas9 nucleases

The Alt-R CRISPR-Cas9 System includes potent Alt-R S.p. Cas9 nucleases. When Alt-R S.p. Cas9 Nuclease 3NLS was combined with the Alt-R CRISPR crRNA and tracrRNA into a ribonucleoprotein (RNP), the system outperformed other editing approaches (Figure 3). You can expect even better editing efficiency with Alt-R S.p. Cas9 Nuclease V3 (see Figure 2). RNP transfections also provide optimal control of dose of editing complexes, and the non-renewable Cas9 RNP is cleared after a short duration by endogenous mechanisms, limiting off-target editing.

Figure 3. Lipofection of Alt-R CRISPR-Cas9 System components as a ribonucleoprotein outperforms other transient CRISPR-Cas9 approaches. Alt-R CRISPR HPRT Control crRNA complexes for human, mouse, or rat were complexed with Alt-R CRISPR tracrRNA. Resulting complexes were transfected with Cas9 expression plasmid, Cas9 mRNA, or as part of a Cas9 RNP (containing Alt-R S.p. Cas9 Nuclease 3NLS, pre-complexed with the crRNA and tracrRNA) into human (HEK-293), mouse (Hepa1-6), or rat (RG2) cell lines. The Cas9 RNP outperformed the other transient Cas9 expression approaches, and performed similar to reference HEK293-Cas9 cells that stably express S. pyogenes Cas9.

The electroporation enhancer is required for efficient genome editing with the CRISPR-Cas12a (Cpf1) system

We have found that some of the Cas12a PAM sequences are not active sites for genome editing (Figure 4). We recommend that you test 3 or more PAM sites in your region of interest and include the Alt-R Cas12a (Cpf1) Electroporation Enhancer for efficient genome editing. The enhancer is a non-targeting carrier DNA that shows no integration into the target site based on next generation sequencing experiments.

User guides and protocols

Improved enzymes: All Alt-R enzymes [Cas9 nuclease, HiFi Cas9 nuclease, Cas9 nickases, and Cas12a (Cpf1) nuclease] have recently been further optimized to deliver even higher performance. The latest versions (V3) can be directly substituted into the protocols in place of the prior Alt-R enzymes.